CH640865A5 - GLYCOPROTEIDE OF HUMAN URINE, METHOD FOR THE PRODUCTION THEREOF AND MEANS FOR COMBATING LEUKOCYTOPENIA. - Google Patents

Description

The invention relates to a glycoproteid, hereinafter referred to as HGI glycoproteid (HGI = human granulocyte inducing), which can be isolated from the urine of healthy people, which acts on the granulopoietic stem cells in the human bone marrow and thereby the proliferation and differentiation of these cells to form Granulocytes stimulated. The invention further relates to a method for obtaining the glycoproteid and medicaments containing this glycoproteid for combating leukocytopenia.

While the peripheral blood of healthy people contains about 5000 to 9000 leukocytes per mm3, the number of leukocytes in leukocytopenia is below about 4000 per mm3. Leukocytopenia is associated with an abnormal decrease in the proliferation of bone marrow cells in some infectious and parasitic diseases, radiation, or administration of cytostatics. For the treatment of leukocytopenia, preparations have been used which contain glycyrrhizin or a mixture of cysteine and glycocoll, a-mercaptopropionylglycine or the alkaloid cepharantine. However, the chemotherapy drugs are undesirable because of their unsatisfactory effects and / or side effects. It has therefore been the goal of numerous research groups to develop drugs for the treatment of leukocytopenia that are more effective and have fewer side effects. It was known that a "colony stimulating lating factor", hereafter referred to as CSF, stimulates the proliferation and differentiation of bone marrow cells. CSF acts on bone marrow cells and io stimulates proliferation and differentiation to form granulocytes or macrophages. It is an essential factor for the bone marrow cells in their in vitro cultivation by simultaneous proliferation and differentiation to form granulocyte or macrophage cell aggregates (hereinafter referred to as granulocyte or macrophage colonies); see. Y. Ichikawa, Proceedings of the National Academy of Science, Vol. 56 (1966), p. 488, and D. Metealf, Expérimental Hematology, Vol. 1 (1973), p. 185. Since CSF prevents the formation of granulocyte and Macrophage colonies 20 induced by bone marrow cells, some research groups proposed to consider CSF as separate factors, namely as granulocyte inducing factor and macrophage inducing factor; see. HE. Stanley et al., J. Exp. Med., Vol. 143 (1976), p. 631. In general, however, these 25 factors are determined together as CSF in vitro tests on mouse bone marrow cells. Numerous factors that stimulate colony formation in vitro in mouse bone marrow cells have been isolated from various sources, e.g., serum, urine, various organ extracts, and media conditioned by different tissues and cell lines, body fluid elements such as serum and urine, conditioned media from Cells such as leukocytes and tissues; see. J.W. Sheridan, Journal of Cell Physiology, Vol. 78 (1971), p. 451. CSF acting on human bone marrow cells 35 was isolated from human sources, namely from serum, various organ extracts and tissue-conditioned media; see. D. Metealf and M.A.S. Moare, “Ciba Foundation Symposium 13, Haemo-poietic Stem Cells”, p. 157, Elsevier Excerpta Medica, Hol-40 land, 1973. However, the CSF obtained from different organs, different cells and their conditioned media is not identical. For example, the molecular weight of CSF from media conditioned by human placenta cells is 30,000 Daltons (see AW Burgess et 45 al., Blood, Vol. 49 (1977), p. 573), while the molecular weight of CSF from human serum is 45,000 daltons (cf. SH Chan et al., British Journal of Haematology, vol. 20 (1971), p. 329). Two types of CSF with a molecular weight of 35,000 and less than 1300 have been isolated from media that have been conditioned by human leukocytes; see. G.B. Price et al., Blood, Vol. 42 (1973), p. 341. The different CSF preparations have a different activity. Some act either on granulocyte cells to be proliferated and differentiated or on macrophage 55 cells. Others act on both types of cells. Therefore, the CSF preparations isolated from different sources are different from each other; see. Metealf and Moore, op. Cit., 1973.

It is also known that a type 60 CSF occurs in human urine which stimulates mouse bone marrow cells to form colonies of granulocytes and macrophages in vitro; see. HE. Stanley et al., Federation Proceedings, Vol. 34 (1975), p. 2272, and E.R. Stanley and D. Metealf, Journal of Experimental Biology and Medicai Science, Vol. 65 47 (1969), p. 467. This CSF preparation is said to have a molecular weight of 45,000 and to proliferate and differentiate mouse bone marrow cells to form predominantly macrophage Stimulate colonies. In contrast

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to its stimulating effect on mouse bone marrow cells, the preparation hardly stimulates the formation of granulocyte or macrophage colonies in human bone marrow cells, but rather stimulates the formation of so-called clusters. In connection with human bone marrow cells, the terms “colony” and “cluster” mean cell aggregates with 40 or more cells or 3 to less than 40 cells as defined by Metealf; see. D. Metealf, Experimental Haematology, Vol. 2 (1974), p. 157.

The invention has for its object to provide a glycoprotein or a CSF preparation and a method for its preparation, which can be used as a drug for combating leukocytopenia. The solution to this problem is based on the finding that an HGI glycoproteid was isolated from human urine, which differs considerably from the known CSF preparations, has a molecular weight of about 85,000 u and the formation in both human bone marrow cells and mouse bone marrow cells pure granulocyte colonies in vitro. The HGI glycoprotein has a strong effect on human bone marrow cells and stimulates the proliferation and differentiation of pure granulocyte colonies. In the following, this effect is sometimes referred to as "biological activity".

Accordingly, the invention relates to a glycoprotein from human urine, which stimulates human bone marrow cells to form granulocyte colonies and which has a molecular weight of 75 to 90,000 u, determined by gel filtration, and has the further properties of claim 1.

The HGI glycoprotein of the invention is prepared as described in the process claims.

The invention is described in more detail below.

A typical process for the preparation of the HGI glyco-proteid of the invention is carried out as follows: Fresh urine from healthy people is adjusted to a pH of 6 to 9, preferably 7 to 8, with dilute acid or base and then for the removal of insoluble ones Centrifuged substances. The supernatant is contacted with a silicon-containing adsorbent such as silica gel, silica gel magnesium silicate, diatomaceous earth, quartz glass or bentonite, and the adsorbed components are eluted with an alkaline solution having a pH of preferably 9 or higher. The type of alkaline solution used for elution is not particularly critical. An aqueous ammonia solution or sodium hydroxide solution is preferably used in a concentration of 0.3 to 1.5M. The eluate obtained is adjusted to a pH of 7 to 8 with an acid and a neutral salt, for example ammonium sulfate up to 70 percent saturation, is added in order to salt out the active ingredient. A crude protein fraction containing the HGI glycoproteid is obtained.

The crude protein fraction obtained is dissolved in a small amount of an alkaline solution, freed from low-molecular substances by ultrafiltration, diluted with a 0.01 to 0.15 molar salt buffer solution and treated with a cation exchanger, for example a carboxymethyl-dextran, carboxymethyl cellulose or phosphocellulose to remove contaminants. The treatment with the exchanger is carried out at neutral pH. The crude fraction of the HGI glycoproteid and the cation exchanger were adjusted to a pH of 6 to 8 with preferably 0.01 to 0.15M salt buffer solution before being brought together. Most of the HGI glycoprotein passes through the cation exchanger without adsorption. The effluent solution is concentrated and the concentrate is equilibrated with a dilute buffer solution of pH 6 to 8 and subjected to ion exchange chromatography on an anion exchanger, such as DEAE cellulose, which has been equilibrated with the same buffer. The HGI glycoprotein is adsorbed on the anion exchanger. The adsorbed HGI glycoproteid is then eluted with a 0.1 to 0.3M saline solution, preferably a sodium chloride solution, with a linear concentration gradient. The HGI glycoprotein is eluted at a salt concentration of 0.1 M or higher. A complete separation is difficult. The fractions of the outflowing solution at a 0.1 to 0.3M salt concentration are collected and optionally desalted and concentrated.

It is also possible to use the stepwise elution with 0.1 to 0.3M saline solution to elute the HGI glycoprotein from the ion exchanger.

For further purification, the combined fractions obtained are subjected to gel filtration chromatography on a strongly crosslinked polymer gel with a water absorption value of 10 to 20 ml per g, for example Sephadex G-150 or Biogel P-100. The active substances are developed with a 0.05 to 0.1 M salt buffer solution. Fractions with a relative elution volume of 1.11 to 1.60, preferably 1.11 to 1.45, are collected, desalted and concentrated or freeze-dried.

The semi-pure HGI glyco-proteid preparations obtained in this way can already be used as drugs.

The relative elution volume is expressed by the quotient Ve / Vo, where Ve represents the elution volume required to elute the substance present in the column and Vo the outer volume (void volume).

For further purification, the semi-purified preparation is dissolved in a dilute salt buffer solution which contains 1.0 to 2.0M, for example a phosphate buffer solution of pH 6.0 to 8.0, preferably 6.0 to 7.0, and Is 1.0 to 2.0M sodium chloride. The solution is subjected to affinity chromatography on an absorbent with affinity for sugar, for example Concanavalin A-Sepharose 4B, which has been equilibrated with the same buffer solution. The HGI glycoproteid adsorbed on the column is mixed with a 1.0 to 2.0M saline solution in a dilute buffer solution, which is 20 to 100 mMolar of saccharides and 1.0 to 2.0M saline, at pH 6.0 to 8 , 0 eluted. For example, the saccharide is an a-methyl-D-glucoside which has a pH of 6.0 to 8.0, preferably 6.0 to 7.0, in solution. The fractions containing the HGI glycoproteid are collected and, if necessary, desalted and concentrated or freeze-dried.

For further purification of the HGI glycoprotein by electrophoresis, the combined fractions from affinity chromatography are subjected to preparative zone electrophoresis using acrylamide gel or agarose gel as a carrier at pH 7.0 to 9.0. Thoroughly purified HGI glycoproteid is eluted from the carrier with dilute salt solution with cooling. The solution obtained is then desalted, concentrated or freeze-dried.

In the course of the process according to the invention for obtaining the HGI glycoproteid, it is possible to simultaneously obtain urokinase, kallikrein and lysozyme.

The highly purified HGI glycoproteid obtained is a white to light brown colored powder, it is tasteless and odorless and somewhat hygroscopic and has the physical and chemical properties described below.

In Fig. 1 the IR absorption spectrum of the HGI-Glyko-

s

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35

40

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50

55

60

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Reproduced proteids. Fig. 2 shows the relationship between the relative mobility in electrophoresis and the molecular weight. FIG. 3 shows the UV absorption spectrum of the HGI glycoproteid and FIG. 4 shows the relationship between the amount of HGI glycoproteid added and the number of colonies which developed in the in vitro test.

The physical and chemical properties were determined on Sample No. 6 from Example 1 (see below).

(1) molecular weight

The molecular weight of the HGI glycoproteid of the invention is about 85,000 u as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 75,000 to 90,000 u as determined by gel filtration on Sephadex G-150. Accordingly, the most reliable molecular weight appears to be between 75,000 and 90,000 Daltons.

Table I (continued)

Acetone chloroform

IM sodium chloride solution 10% sucrose solution insoluble slightly soluble soluble soluble

In addition, HGI glycoproteide is easily soluble in dilute salt solutions, for example in a dilute phosphate solution or a dilute trisaminomethane solution. It is also soluble in a dilute saline solution in the pH range of 1 to 12.

15 (3) pH

The pH of a 1 percent aqueous solution of the HGI glycoproteid is 5.0 to 6.0. So it reacts weakly acidic.

(2) solubility

The solubility of the HGI glycoprotein in various solvents is summarized in Table I.

Table I

solvent

solubility

Water ethanol insoluble

20 (4) Specific rotation value

The specific rotation value is determined on a 0.25 percent aqueous solution of the HGI glycoprotein at 20 ° C. and the wavelength of the D line. [oc] d = 0 ± 40 °.

25 (5) IR absorption spectrum

The IR absorption spectrum of the HGI glycoprotein as a potassium bromide compact is shown in FIG. 1. The characteristic absorption bands are summarized in Table II.

Table!!

Wavenumber, cm "1

absorption

Remarks

3600-

-3200

strong the broad absorption band apparently comes from the v-OH groups,

which form hydrogen bonds to varying degrees

1700-

-1600

strong \

the broad absorption band apparently comes from the

1550

medium /

-CO-NH bonds of the protein

1430-

-1380

medium

1150-

-1000

medium the broad absorption band apparently comes from the -C-O-C-

Polysaccharide bonds.

(6) Isoelectric point

The isoelectric point of the HGI glycoprotein is at pH 4.7 ± 0.2, determined by polyacrylamide gel electrofocusing.

45

(7) color reactions

Color reactions of the HGI glycoprotein are carried out in aqueous solution. The results are summarized in Table III.

Table III

Color reaction

colour

Remarks

Lowry process *

Ninhydrin (hydrolysis with 6N HCl at 110 ° C for 22 hours)

a-Naphthol-sulfuric acid (Molisch)

Indole-sulfuric acid (Dische)

Anthrone-sulfuric acid

Phenol-sulfuric acid blue violet violet brown dark green brown

Peptide bonds a-amino acids

Saccharides

Saccharides

Saccharides

Saccharides

■ * Edited by Sidny P. Colowick and Nathan O. Kaplan, "Methods in Enzymology", Vol. III, page448 (Academic Press Inc., Publishers, New York, 1957) ... (Item 9)

(8) Thermostability 65 (9) Amino acid composition of the protein fragment When heating a 1% aqueous solution for 30 minutes ProActivity amino acid composition no longer ascertainable. fragment is determined using an amino acid auto-analysis

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determined. The results are summarized in Table IV.

Table IV

amino acid

% By weight

Mole (mmole)

Proline

3.2

0.392

Aspartic acid

9.8

1,038

Threonine

2.8

0.331

Serine

11.9

1,596

Glutamic acid

13.8

1,322

Glycol

11.0

2,066

Alanine

7.3

1,155

Valine

6.4

0.771

Methionine

2.5

0.236

Isoleucine

2.5

0.269

Leucine

7.0

0.753

Tyrosine

5.8

0.451

Phenylalanine

12.8

1,050

Lysine

2.2

0.212

Histidine

1.0

0.091

Tryptophan

traces

-

Arginine

traces

-

ammonia

0.5

-

From Table IV it can be seen that the protein fragment of the HGI glycoprotein consists of 17 amino acids, the acidic and neutral amino acids predominating, while the basic amino acids are present in minor amounts. It is also characteristic that over 70% of the total amino acids are unbranched amino acids, including aspartic acid, threonine, serine, glutamic acid, glycocoll, alanine, valine and leucine.

(10) electrophoresis

According to the method of Laemuli, Nature, Vol. 227 (1970), p. 680, and using sodium dodecyl sulfate-polyacrylamide gel, the HGI glycoproteid shows a single band at a position of a relative mobility of 0.25. At the same time, the trypsin inhibitor (molecular weight 21,500), ovalbumin (molecular weight 43,000), monomeric human serum albumin (molecular weight 65,000) and dimeric human serum albumin (molecular weight 130,000) were subjected to electrophoresis. From the mobility of the known molecular weight substances and the mobility of the HGI glycoproteid, the molecular weight of the glycoproteide is calculated to be approximately 85,000 (FIG. 2). In Fig. 2, the letters a, b, c and d mean the trypsin inhibitor, ovalbumin, monomeric human serum albumin and dimeric human serum albumin. The arrow indicates the HGI glycoprotein.

(11) UV absorption spectrum

The UV absorption spectrum of the HGI glycoprotein, determined on a 0.1 percent aqueous solution in a quartz cell with a layer thickness of 1 cm, is shown in FIG. 3. The absorption spectrum shows an absorption maximum at 280 nm and final absorption in the wavelength range below 250 nm. The optical density Ej ^ at 280 nm is 3.8.

(12) Sugar composition of the polysaccharide fragment

Neutral sugars were obtained by the phenol-sulfuric acid reaction sialic acids according to the thiobarbital method of Warren [Journal of Biological Chemistry, Vol. 234 (1959), p. 1971] and amino sugar according to the method of Elson-

Morgan [Biochemical Journal, Vol. 27 (1933), p. 1824]. The amount of neutral sugar is expressed in glucose. The following results were obtained: neutral sugar 10.0 to 13.0%; Sialic acids 3.0 to 7.0%; Aminosugar less than 1.0%; Total sugar 13.0 to 20.0%.

(13) Protein and polysaccharide content

The protein content of the HGI glycoprotein is 75 to 85%, determined according to the semi-micro Kjeldahl method. The total sugar content is 13.0 to 20.0% as described above.

(14) Elemental analysis

The elementary analysis of the HGI glycoprotein shows the following values: carbon 42.3 to 47.3%; Hydrogen 5.7 to 7.8%; Nitrogen 9.6 to 14.3%; Oxygen 34.4 to 39.4%; Sulfur less than 0.2%.

The HGI glycoprotein of the invention having the above physical and chemical properties stimulates the proliferation and differentiation of both human and mouse granulocytes. This can be seen from Experiment 1 described below. The glycoprotein shows no acute toxicity, as can be seen from Experiment 4 described below. It can be seen from experiment 3 described below that the glycoproteid can be used to combat leukocytopenia.

The HGI glycoproteid of the invention made from human urine by the method described above is freeze-dried in vials under aseptic conditions and then hermetically sealed. Before freeze-drying, it is possible to add an aqueous solution of human serum albumin as a stabilizer and an amino acid or a saccharide to improve the solubility of the HGI glycoproteid. The solution obtained is sterilized by membrane filtration and then freeze-dried aseptically. Before use, the vessel is opened and the HGI glycoproteid is dissolved by adding sterile physiological saline, sterile water or a sterile isotonic solution. The solution obtained can be injected intravenously, intramuscularly or subcutaneously.

The results of experiments 1 and 2 described below show that the effective daily dose is at least 0.75 mg, preferably 0.75 to 2.24 mg, per kg of body weight. Semi-purified, large-scale preparations with a specific biological activity of at least about 35,000 units per mg, for example the HGI glycoproteid sample No. 4 and No. 5 from Example 1, can also be used as drugs.

The effect of the HGI glycoprotein of the invention on the proliferation and differentiation of granulocytes is described in detail below.

Trial 1

Stimulating effect on the proliferation and differentiation of mouse and human granulocytes in vitro

Plastic petri dishes with a diameter of 35 mm are each charged with 1 ml of McCoy's 5 A medium containing 0.05, 0, 1, 15 and 0.2 pg of HGI glycoproteid (Sample No. 6 from Example 1) , 20% fetal calf serum, 0.3% agar and 7.5 x 104 mouse bone marrow cells or 25 x 104 bone marrow cells from healthy people or patients with iron deficiency anemia. The medium in the Petri dishes is incubated in a humid, 5% CO2 atmosphere for 7 to 9 days at 37 ° C. The difference in the number of vaccinated mouse cells and human cells is due to the

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larger number of stem cells in the case of the mouse. After incubation, single colonies containing more than 50 mouse cells or more than 40 human cells are counted with an inverted microscope (inverted beam path). For the morphological analysis of the colonies, some of them are inoculated in microhaematocrit tubes and stained with 0.6 percent orcein in 40 percent acetic acid. The results are shown in FIG. 4. The figure shows the relationship between the HGI glycoproteid dose and the number of colonies formed in vitro. In Fig. 4, the circles represent the mouse bone marrow cells and the dots the human bone marrow cells.

It can be seen from Fig. 4 that the HGI glycoproteid of the invention stimulates the proliferation and differentiation of mouse and human bone marrow cells. There is a dose-response relationship between the HGI glycoproteid and the number of colonies formed.

The morphological analysis of the cells in the colonies shows that all cells are mature granulocytes.

As described above, the HGI glycoproteid acts on human and mouse bone marrow cells to form granulocyte colonies, the number of colonies being proportional to the dose of the HGI glycoproteid. There is a certain relationship between the formation of mouse and human bone marrow cell colonies. Therefore, only bone marrow cells from the mouse were used in all subsequent experiments.

Trial 2

Stimulating effect on the proliferation and differentiation of granulocytes in vivo

60 male mice of the Cs7-BL strain with an average body weight of 20 g are statistically divided into 6 groups of 10 animals each. One group was used for comparison. This group is given subcutaneously 0.04 mg per mouse of human serum albumin in 0.2 ml of sterile normal saline once a day for three consecutive days. The remaining 5 groups are subcutaneously 0.005, 0.01, 0.02, 0.03 or 0.04 mg per mouse HGI glycoproteid of sample No. 6 from Example 1, dissolved in 0.2 ml of 15 sterile normal saline, given once a day for three consecutive days.

Blood samples are taken from the coccygeal vein of each mouse before injection and 2,4,6, 8 and 10 days after injection. The leukocytes of each blood sample are stained with 1% Gentiana violet solution and the leukocyte count is counted in a Bürker-Türk counting chamber.

Furthermore, each blood sample is spread on a slide, stained with Wright-Giemsa solution, and the proportion of granulocytes in the leukocytes is determined microscopically.

The number of granulocytes is calculated using the following equation:

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Leukocyte count in 1 mm3 x number of granulocytes in the leukocytes = number of granulocytes in 1 mm3 The results are summarized in Table V.

Table V

Group comparison group 1 2 3 4 5

Dose, mg

Days 0 0.005 0.01 0.02 0.03 0.04

0 450 452 455 450 448 445

2,380,420,550,600,775,800

4 372 390 800 1100 1400 1420

6 380 395 1100 1800 2200 2100

8 410 400 620 750 980 971

10 430 410 470 460 465 480

Note: The numerical values represent the number average of the granulocytes per mm for 10 mice.

From Table V it can be seen that the peripheral granulocyte count of the test groups given 0.01 to 0.04 mg per mouse of HGI glycoproteid increased two days after the administration and after 6 days the 3- to 6-fold value of the comparison group reached.

The number of granulocytes then decreases and after 10 days the normal value is reached again. When the daily dose was increased to more than 0.04 mg, there was no significant increase in the granulocytes corresponding to the dose of the HGI glycoproteid.

These results show that by injecting at least 0.01 mg, preferably 0.01 to 0.03 mg, of HGI-Gly-coproteid into a mouse with an average body weight of about 20 g of granulocytosis daily. Since the stimulating effects of the HGI glycoprotein on mouse bone marrow cells in vitro are on average about 1.5 times higher than the effect on human bone marrow cells (experiment l), the effective dose for humans is 1.5 times as high as for the mouse, determined in vivo after experiment 2. Accordingly, the effective daily dose in humans per kg of body weight is at least about 0.75 mg, preferably 0.75 to 2.24 mg.

Trial 3

Protective effect of the HGI glycoprotein caused by cytostatic leukocytopenia 30 male, 4 to 5 week old mice of the Cs7BL strain are statistically divided into three groups of 10 animals each. The control group is given intraperetoneally 30 mg per kg body weight (corresponds to 1/10 LDso) cytosine-D-ara-binoside, dissolved in 0.2 ml of sterile normal saline 55 once a day for 14 consecutive days. In addition, 0.2 ml per mouse of sterile normal saline is given subcutaneously once a day for 14 consecutive days. The first test group is given cytosine D-arabinoside like the comparison group 60. In addition, 0.03 mg per mouse of HGI glyco-proteid of Sample No. 6 of Example 1 is given subcutaneously once a day for 14 consecutive days. The remaining test group is also given cytosine D-arabinoside as the comparison group and subcutaneously 65 0.3 mg per mouse cepharantin, dissolved in 0.2 ml of sterile normal saline, once a day for 14 consecutive days.

Blood samples are taken from the coccygeal vein of each mouse before

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the administration and on the 2nd, 4th, 6th, 8th, 10th, 12th and 14th day is followed. The number of leukocytes before administration

taken after administration. The leukocyte count is assumed to be 100. The results are determined in the same way as in experiment 2 and the percentage table VI is compiled.

Decrease (decrement) in the number of leukocytes after administration

Table VI

Group comparison groups HGI glycoproteid group cepharantin group

Days after leukocyte count decrement (%) leukocyte count decrement (%) leukocyte count decrement (0 / o)

Administration (xl02 / mm3) (xl02 / mm3) (xloVmm3)

before the

administration

150

100

150

100

150

100

2nd

130

86.6

120

80.0

125

83.3

4th

125

83.3

125

83.3

120

80.0

6

120

80.0

130

86.6

120

80.0

8th

110

73.3

120

80.0

110

73.3

10th

95

63.3

120

80.0

100

66.6

12

80

53.3

115

76.6

105

70.0

14

70

46.6

110

73.3

95

63.3

Note: Each numerical value is the average for 10 mice.

In comparison to the comparison group, the HGI glycoprotein group shows a pronounced protective effect against the reduction in the number of leukocytes 10 days after the start of the HGI glycoprotein administration. The effect is at least as good as that of cepharantine. On the 14th day after the start of treatment, the leukocyte count of the comparison group was reduced to 46.6%, while that of the HGI glycoproteid group was 73.3%. The reduction is less than that of the cepharantin group. From these values it can be seen that the HGI glycoproteid is effective for the therapy of human leukocytopenia.

Further experiments have shown that the HGI glycoproteid of the invention is also effective against other cytostatics, for example 5-fluorouracil and daunomycin, which cause a reduction in the number of leukocytes similar to the cytosine-D-ara-binoside. Human serum albumin has no preventive effect in reducing the number of leukocytes under the same experimental conditions.

Trial 4

Acute toxicity of the HGI glycoprotein

The acute toxicity of the HGI glycoproteid, produced according to Example 1, Sample No. 4 and No. 6, is determined on male mice of the C57BL strain according to the method of Litch-field and Wilcoxon [Journal of Pharmacology and Experimental Therapeutics, Vol. 90 (1949), p. 99]. The glycoproteid is administered intraperitoneally at a dose of 4000 mg per kg body weight or 2000 mg per kg body weight intravenously. As a result, it was practically impossible to estimate the LDs. The LDso is above 4000 mg per kg for subcutaneous injection and above 2000 mg per kg for intravenous injection.

example 1

400 liters of fresh urine from healthy people is adjusted to a pH of 8 with 10% sodium hydroxide solution and centrifuged in a continuous centrifuge at 15,000 x g and 0 ° C to remove insoluble substances. The supernatant is adjusted to pH 7 with 10% hydrochloric acid and placed on a column filled with silica gel with the dimensions 10 × 80 cm. The substances adsorbed on the silica gel are eluted with 40 liters of 5 percent ammonia solution. The eluate obtained is adjusted to pH 7.5 with sulfuric acid and ammonium sulfate is added to a saturation level of up to 70 percent. The solution is then left to stand at 0 ° C. for 15 to 18 hours. The resulting precipitate is filtered off, dissolved in 2 liters of 5 percent aqueous ammonia solution, filled into cellophane tubes and dialyzed against 0.05M phosphate buffer solution with a pH of 6.5. The retentate is made up to 10 liters with the same buffer solution and placed on a column filled with CM Sephadex C-50 with the dimensions 40x40 cm. This column had been equilibrated with 0.05M phosphate buffer pH 6.5. Impurities are adsorbed on the ion exchanger. 10 liters of the elution solution are concentrated using a DIAFLO hollow fiber ultrafiltration device (Amicon DC-30; molecular weight cut-off limit about 10,000). The concentrated solution is dialyzed against 0.1M tris-HCl buffer solution of pH 7.0 at 5 ° C. for 15 to 18 hours. The retentate is made up to 1 liter with the same buffer solution. The solution obtained is referred to as Sample No. 1.

The solution obtained is passed through a DEAE cellulose column with the dimensions 4.0 × 40 cm, which had previously been equilibrated with 0.1 M tris-HCl buffer with a pH of 7.0. The column is then washed with a sufficient volume of 0.1M tris-HCl buffer solution of pH 7.0. Then, elution is carried out in stages with 0.1 M tris-HCl buffer solution of pH 7.0, which is 0.3 M sodium chloride. The fractions which cause proliferation and differentiation of granulocytes, as described in experiment 1, are collected and dialyzed against 0.1 M tris-HCl buffer solution with a pH of 7.0. This solution is referred to as Sample No. 2.

The dialyzed solution (the retentate) is again placed on a DEAE cellulose-filled column with the dimensions 4.0 × 40 cm, which had been equilibrated with 0.1 M tris-HCl buffer solution with a pH of 7.0. The column is then eluted with a linear concentration gradient from 0 to 0.3 M saline. The active fractions containing the glycoproteid are collected and ammonium sulfate is added to 70 percent saturation. The resulting precipitates are centrifuged off and dissolved in a small volume of 0.1M tris-HCl buffer solution with a pH of 65 7.0 and dialyzed against the same buffer solution. This dialyzed solution (retentate) is referred to as Sample No. 3.

20 ml of the dialyzed solution are placed on a column of dimensions filled with Sephadex G-l 50

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4.0x60 cm, which had been equilibrated with 0.1 M tris-HCl buffer solution with pH 7.0. The elution fractions with a Ve / Vo ratio of 1.11 to 1.45 are collected. The combined fractions are dialyzed thoroughly against distilled water at 5 ° C. The dialyzed solution (retentate) is then freeze-dried. About 500 mg of a powder are obtained. This semi-purified HGI glycoproteid is designated Sample No. 4.

200 mg of the semi-purified HGI glycoprotein are dissolved in 0.02 M phosphate buffer solution with a pH of 7.0, which is 1.0 M in sodium chloride, and placed on a column filled with 100 ml of Conca-navalin A-Sepharose 4B, the with the same buffer solution had been equilibrated. After washing the column thoroughly with the same buffer, the HGI glycoproteid is eluted with 0.02M phosphate buffer solution of pH 7.0, which is 50mM of a-methyl-D-glucoside and 1.0M of sodium chloride. Those fractions which cause granulocyte proliferation and differentiation in vitro are collected and dialyzed against distilled water. The dialyzed solution (retentate) is freeze-dried. This preparation is referred to as Sample No. 5.

About 50 mg of the freeze-dried powder obtained are dissolved in 1 ml of a 0.125 M tris-glycocoll buffer solution of pH 6.8, which contains 10% glycerol. The solution obtained is subjected to electrophoresis at 10 mA with cooling using a preparative electrophoresis device (Fuji Kabara II type) using 8% acrylamide gel (pH 8.9; 20 mm × 25 mm). The fraction with a relative mobility of 0.46 is isolated with 0.025 M tris-glycocoll buffer of pH 8.3 and dialyzed against distilled water. The dialyzed solution (retentate) is freeze-dried. About 10 mg of the HGI-Gly-coproteid are obtained, which is referred to as sample No. 6, io. The sample numbers 1 to 6 obtained in the various production stages are determined according to the proliferation and differentiation of the bone marrow cells of both humans and mice Experiment 1 examined. The HGI glycoproteid or the preparation containing this glycoproteid is added to the medium in such an amount that 200 colonies form per petri dish. The specific activity is calculated according to the following equation, where one unit corresponds to a colony formed.

20 Specific activity (units / mg)

Number of colonies formed (units)

Protein content (mg) of the tested sample

25 The results are summarized in Table VII.

Table VII

Mouse Bone Marrow Cell Test Human Bone Marrow Cell Test

Sample specific activity subjects cleaning level Specific activity subjects cleaning level

(Units / mg) (units / mg)

No. 1 260 1 160 1

No. 2 4 500 17.3 2 200 13.8

No. 3 11,000 42.3 5 500 34.4

No. 4 57,000 219.2 35,000 218.8

No. 5 210 000 807.7 180 000 1125.0

No. 6 1 000 000 3846.1 670000 4187.5

Example 2

100 mg of the HGI glycoproteid powder (sample No. 6 from Example 1) are mixed with 100 ml of an aqueous solution which contains 5% human serum albumin and 1% glycoll. The solution obtained is sterilized by filtration using membrane filters with a pore size of 0.45 ji. The sterilized solution is then filled aseptically in 1 ml portions into vessels which have been sterilized beforehand by heating at 180 ° C. for two hours. After freeze-drying, the vessels are hermetically sealed. In this way, 95 ampoules of a drug for the treatment of leukocytopenia are obtained. Each ampoule contains 1 mg HGI glycoproteid.

Example 3

In the manner described in Example 1, 1 liter of a concentrated solution containing HGI glycoproteid is prepared (analogously to sample No. 1), namely from 1000 liters of fresh urine from healthy people. This concentrated solution is diluted with 10 liters of a 0.1M tris-HCl buffer solution with a pH of 7.0. After thorough stirring, the diluted solution is concentrated to about> / io of its original volume using a DIAFLO hollow fiber ultrafiltration device. The concentrated solution obtained is mixed with 5 liters of a 0.1M tris-HCl buffer solution with a pH of 7.0 and 5 liters of a DEAE cellulose suspension which contains 300 g of DEAE cellulose (calculated on a dry basis) and which contains 0. 1M pH tris-HCl buffer solution

7.0 had been equilibrated. The mixture is stirred for 30 minutes, then left to stand for 10 minutes and finally filtered on a Buchner funnel under reduced pressure. DEAE cellulose is obtained. The filtered-off DEAE cellulose is then washed with 10 liters of a 0.1 M tris-HCl buffer solution with a pH of 7.0 and filtered off again on the Buchner funnel. The DEAE cellulose is then washed with 10 liters of a 0.1 M tris-HCl buffer solution with a pH of 7.0, which is 0.05 M in sodium chloride, and is filtered off in the same way on the Buchner funnel. The DEAE cellulose treated in this way is mixed with 10 liters of a 0.1 M tris-HCl buffer solution of pH 7.0, which is 0.3 M sodium chloride, and stirred to remove the HGI glycoproteid from the DEAE -Cellulose to separate. The SS mixture is filtered off on a Büchner funnel and the filtrate is desalted by DIAFLO hollow fiber ultrafiltration. The desalted solution obtained is freeze-dried. About 15 g of a powder are obtained. The powder is dissolved in 150 ml of distilled water and placed on a 6.0 x 80 cm column filled with Sephadex so G-150, which had been equilibrated with 0.1M tris-HCl buffer solution with a pH of 7.0 . The fractions are collected according to the Ve / Vo ratio of 1.11 to 1.60. The combined fractions are dialyzed thoroughly against distilled water. The dialyzed solution (retentate) is then concentrated using a DIAFLO hollow fiber ultrafiltration device (type DC-2) to about 100 ml of concentrate which contains about 3 g of crude HGI glycoproteide. The

640 865

concentrated solution is washed with 1 g glycocoll and 5 g serum albumin. The solution obtained is sterilized according to Example 1 by filtration and then filled aseptically in 2.5 ml portions into ampoules. After aseptic freeze drying, the ampoules are hermetically sealed. 40 ampoules of a drug to combat leukocytopenia are obtained. Each ampoule contains approximately 3.8 mg HGI glycoproteid.

For clinical use, it is important to separate pathogenic viruses that are contained in the urine and that cause hepatitis and other diseases from the HGI glycoprotein. This is done by heat inactivation analogous to the heat inactivation of the viruses in serum albumin by heating at 60 ° C. for 10 hours. To carry out heat inactivation at 60 ° C for 10 hours, the substances to be treated must be stable under these conditions. Various stabilizers have been developed for this purpose. Various amino acids or saccharides with physiologically isotonic concentrate or below are used to stabilize human serum proteins against heat. When viruses are thermally inactivated in HGI glycoproteid, the biological activity of the glycoproteid is lost by heating to 60 ° C. for 10 hours.

It was found that the heat stability of the HGI glycoproteid in aqueous solution can be improved under the following conditions and its characteristic properties are not lost. The HGI glycoprotein is stable against heat treatment at pH 5 to 9 if albumin is in the solution in a concentration of at least 2% or the concentration of the HGI glycoprotein in the solution is at least 70 mg per ml, or if human proteins from the urine in a concentration of at least 70 mg per ml.

The invention thus provides an HGI glycoprotein which can be subjected to thermal inactivation in order to inactivate viruses. This thermal inactivation consists in heating an aqueous solution of the HGI glycoproteid, which is heated to 50 to 70 ° C., preferably 55 to 65 ° C. for 8 to 30 hours, preferably 8 to 12 hours and at a pH of 5 to 9 has been stabilized.

When using albumin as a stabilizer, the degree of purification of the HGI glycoprotein contained in the aqueous solution is not critical, but HGI glycoprotein fractions of a purity corresponding to those of samples No. 4 to No. 6 of Example 1 are preferred.

The concentration of the HGI glycoprotein in the aqueous solution to be subjected to the heat treatment is preferably at least 0.1% (weight per volume, ie number of weight units in 100 volume units; the same applies to the aqueous solution below), preferably 0.5 to 15%. The aqueous solution is preferably adjusted to a pH of 5 to 9, preferably 6 to 8, with an acid or base, preferably a buffer solution. The albumin preparation used can come from human serum or human placenta. Both preparations have preferably been cleaned for medical purposes and have a purity of at least 80%, determined by electrophoresis. The amount of albumin added to the aqueous HGI glycoproteid solution is normally at least 2.0, preferably 10 to 20%. The upper limit of the amount of albumin added is not particularly critical, and the appropriate amount depends on the permitted amount of albumin in the final product.

When using human albumin to stabilize the HGI glycoproteid against heat, there is no risk of contamination of the end product with antigenic substances, and an effective stabilization of the glycoproteid against heat inactivation is achieved. Since albumin also ensures the stability of the glycoprotein preparations, it is not necessary to separate the albumin after the heat treatment, provided that a suitable amount is used in the heat treatment. Therefore, the heat treatment according to the method of the invention is a preferred embodiment for the production of a non-infectious HGI glycoproteid.

Trial 5

The effect of heat treatment in the presence of albumin on viral infectivity is being investigated using various viruses that may contaminate HGI glycoproteid preparations.

The HGI glycoprotein sample No. 4 of Example 1 is dissolved in water in an amount of 100 mg per ml. This solution is mixed with 0.2 ml of a virus suspension with a virus-infectious dose of 1 x 105 to 1 x 106 per 0.2 ml (measured according to the method described below) pox virus, mumps virus, measles virus, herpes virus, chikungunya virus, Japanese encephalitis virus, rubella virus, Poliovirus, Coxsackievirus or ECHO virus and then added 0.20, 100 or 200 mg per ml of human serum albumin. The solution obtained is adjusted to a pH of 7.0 with 0.1M tris-HCl buffer solution and then heated to 60 ° C. for 10 hours, then immediately cooled in ice-cold water, desalted and sterilized by membrane filtration and finally freeze-dried. Biological activity and viral infection are determined according to the methods described below and compared with the values obtained before the heat treatment.

The biological activity is determined on the basis of the colony formation of mouse bone marrow cells in vitro. Petri-plastic dishes with a diameter of 35 mm are mixed with 1.0 ml of supplemented McCoy 5 A medium, the 20% fetal calf serum, 10% (0.1 ml) sample, 0.3% agar, 7.5 x 104 mouse bone marrow cells and contains 10% (0.1 ml) of the sample. The concentration of each sample is set to the same value. After 7 days of incubation in a moist 5 percent carbon dioxide atmosphere, single colonies with more than 50 cells are counted with the inverted microscope. The percentage ratio of the number of colonies formed after the heat treatment to the number of colonies before the heat treatment is called the recovery of biological activity.

The virus-infectious dose is determined as follows: smallpox virus is inoculated onto HeLa cells and the plaque-forming units [Virology, Vol. 36 (1968), pp. 174 to 179] are determined with the same cells. Subsequent grafts of mumps virus, measles virus and Japanese encephalitis virus are performed with Vero cells. Those with poliovirus, coxsackievirus and ECHO virus are carried out with HeLa cells. The infectivity of each virus is determined by determining the 50 percent infectious dose for tissue culture (National Institute of Health, Japan, "Experimental Virology", Maruzen Co., Tokyo, 1967). The cytopathic effect is used as an indicator. The observation period is 10 days.

Herpes virus and chikungunya virus are vaccinated in succession in FL cells and Vero cells. The PIaques-forming units are then determined.

Rubella virus is sequentially vaccinated in BHK-21 cells. The PIaques-forming units are determined on Vero cells. The results are summarized in Table VIII.

10th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

11

Vili table

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Concentration of human serum albumin before heating

(mg / ml) number of colonies per virus infection

0.1 ml

After heating the biological recovery

Number of colonies per virus infection activity (%) 0.1 ml

200 116 yes

100 124 yes

20 118 yes

0 (comparison) 121 yes

128 no 110.3

134 no 108.1

51 no 43.2

0 no 0

From Table VIII it can be seen that after heating at 60 ° C for 10 hours, the viral infectivity was completely lost in each sample, regardless of whether albumin was added or not. This shows that other viruses can also be inactivated by heat treatment according to the method of the invention. The biological activity of the HGI glycoprotein was found to be maintained in the samples containing albumin. This shows that the albumin is an effective stabilizer.

The results of the heat treatment under different conditions are similar to those mentioned above.

In the next experiment, the pH of the aqueous albumin solution containing HGI glycoproteid is varied to determine the change in biological activity after the heat treatment.

Trial 6

The HGI glycoproteid used in experiment 5 is dissolved in water to a solution with a concentration of 20 mg per ml. The solution obtained is mixed with 100 mg per ml of human serum albumin. Solutions with a pH of 2 to 10 with intervals of 1 unit are prepared as follows: pH 2 to 6.0.1 M citric acid sodium phosphate buffer, pH 7 to 1Ó, 0.1M tris -HCl buffer. Each sample is heated to 60 ° C for 10 hours. The remaining biological activity is then determined in accordance with experiment 5. The percentage ratio of the number of colonies formed after the heat treatment to the number of colonies before the heat treatment is called the recovery of biological activity. The results are summarized in Table IX. The biological activity of the HGI glycoprotein is well preserved in the pH range from 5 to 9. This shows that it is necessary to adjust the pH of the HGI glycoproteid solution to this range before the heat treatment.

Table IX

pH number of colonies recovery per 0.1 ml of biological activity before

86

100.0

Heat treatment

2nd

0

0

3rd

0

0

4th

0

0

5

82

95.3

6

96

111.6

7

118

137.2

8th

100

116.3

9

68

79.1

10th

0

0

In an experiment described later, an aqueous

Solution containing at least 70 mg per ml of HGI glycoproteide is adjusted to a pH of 2 to 10 with an acid or base, and the recovery of the biological activity after the heat treatment is determined. It has been found that the recovery of biological activity is about 40 to 50% at a pH in the range of 5 to 9, while the biological activity 20 of an aqueous solution of the HGI glycoproteid is at a pH below 5 or above 9 has completely disappeared after the heat treatment.

Trial 7

25 The dependence of the effect of the heat treatment on the concentration of the HGI glycoprotein in the aqueous solution is examined.

The purified HGI glycoproteid from Sample No. 6 of Example 1 is dissolved in water and adjusted to pH 7.0 with 0.1M tris-HCl buffer solution. The solution is then adjusted to final concentrations of 10.50, 60, 70, 80, 100, 150 and 200 mg per ml. Each solution is heated to 60 ° C for 10 hours, then quickly cooled in ice water, sterilized by filtration and freeze-dried. The biological activity of each sample is determined according to experiment 5. The results are summarized in Table X.

Table X

40

Concentration of the number of colonies recovering the

HGI glycoprotein per 0.1m! biological activity

(mg / ml)

10th

0

0

50

31

25.4

60

35

28.2

70

44

35.5

80

50

40.3

100

51

41.1

150

53

42.7

200

56

45.2

before the

124

100.0

Heat treatment

From Table X it can be seen that at a concentration of the HGI glycoproteid of 50 mg per ml, the recovery of the biological activity after the heat treatment is 25.4%. To recover a biological activity of at least 35% after the heat treatment, the concentration of the HGI glycoprotein must be at least 70 mg per ml, preferably 100 to 200 mg per ml.

Trial 8

65 The HGI glycoproteid used in experiment 5 is dissolved in water and various pH values in the range from 2 to 10 are set. For the pH range from 2 to 6, 0.01M citric acid sodium phosphate buffer solution and for the

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12

pH range from 7 to 10 0.1 M tris-HCl buffer solution used. Each solution is adjusted to a final concentration of 100 mg per ml with distilled water and heated to 60 ° C. for 10 hours, then quickly cooled in ice water and tested for biological activity according to experiment 5. Part of the solution (pH 4.5) before adjusting the pH is used as a reference. The results are summarized in Table XI.

Table XI

PH value

Number of colonies per 0.1 ml

Recovery of biological activity (%)

before the

136

100

Heat treatment 2

0

0

3rd

0

0

4th

0

0

5

51

37.5

6

54

39.7

7

59

43.3

8th

63

46.3

9

49

36.0

10th

0

0

pH not adjusted

0

0

From Table XI it can be seen that at a pH of the HGI glycoproteid solution in the range from 5 to 9, the recovery of the biological activity is 36.0 to 46.3%. At a pH below 5 or above 9, the biological activity is completely lost. The results of experiments 7 and 8 show that the heat treatment of the HGI glycoproteid solution means that the concentration of the glycoproteid is at least 70 mg per ml and the pH should be in the range from 5 to 9.

Trial 9

Experiment 5 is repeated, but the HGI glycoproteid is dissolved in water at a concentration of 200 mg per ml. No serum albumin is added and the solution is adjusted to pH 7.0. After the heat treatment, only the virus-infectious dose is determined, as in experiment 5. In this example, virus activity has been completely lost. The result shows that viruses other than those used can also be inactivated by the heat treatment using the method according to the invention.

The above-described method of virus inactivation by heating in the presence of a stabilizer is applied to HGI glycoproteid preparations with a certain degree of purity. Therefore, a practical process was sought that can be used directly to produce a purified HGI glycoproteid. In the course of these investigations, it was found that the HGI glycoproteid solution, which contains at least 70 mg per ml of a protein from the urine used as the starting material, is a heat-resistant solution for this purpose. This new method for stabilizing the HGI-Gly-coproteid solution against heating is described in more detail below.

1 liter of fresh urine from healthy people usually contains 30 to 50 mg of protein. These urine proteins are concentrated by ultrafiltration, ion exchange chromatography, adsorption on silica gel, salting out or a combination of these methods. The proteins from urine, including the HGI glycoprotein, will be reduced! Pressure evaporated further to bring the protein content to at least 70, preferably 100 to 150 mg per ml. If the protein content of the fraction containing HGI glycoproteid is less than 70 mg per ml, the biological activity of HGI glycoproteid 20 drops very sharply when heated. Although each crude fraction containing 70 mg per ml or more of urine proteins serves the purpose, a crude fraction obtained in the above-described method for producing the HGI glycoproteid of the invention is preferably used for the new stabilization method. The crude fraction is adjusted to a urine protein content of at least 70 mg per ml and a pH of 5 to 9, preferably 6 to 8.

The heat-stabilized HGI glycoproteid solution obtained is optionally subjected to heat treatment under the conditions described above after purification by the method described above, and a fraction is obtained which can be used directly as a medicament.

35 Trial 10

Example 1 is repeated. A fraction containing HGI glycoproteid is obtained analogously to sample No. 1, with the exception of the following modification:

The precipitate obtained by salting out with ammonium sulfate up to 70% -40% saturation is divided into 8 parts. Each part is dissolved in water and adjusted to pH 7.0 with 0.1M tris-HCl buffer solution. The 8 solutions obtained are adjusted to a protein content of 10.50, 60, 80, 100, 150 or 200 mg per ml. 45 Each solution is further divided into two groups. One group is heated to 60 ° C for 10 hours, the other remains untreated.

All solution samples are further purified according to Example 1 except for the degree of purification of Sample No. 4 as in Example 1. Two rows of samples with different protein contents are obtained in this way. One row (test samples) is heat treated, the other row (comparison samples) is untreated. Each sample is examined for its biological activity according to experiment 5. The results are summarized in Table XII.

Table XII

Sample No. Protein content (mg / ml) number of colonies per 0.1 ml recovery of biological

Activity ("■»)

Comparative sample 1 10 106

2 50 111

3 60 115

4 70 106

5 80 110

6 100 118

7 150 120

8 200 137

13 640865

Table XII (continued)

Sample No. Protein content (mg / ml) number of colonies per 0.1 ml recovery of biological

Activity (%)

Test sample 9 10 45 42.5

10 50 89 80.2

11 60 98 85.2

12 70 104 98.1

13 80 109 99.1

14 100 121 102.5

15 150 123 102.5

16 200 141 102.9

Example 4 15

The procedure according to Example 1 is repeated several times to produce 100 mg of HGI glycoproteid corresponding to the degree of purity of sample No. 6,100 mg of the purified glycoproteid obtained are mixed with 100 ml of 10 percent human serum protein as a stabilizer. The solution obtained is adjusted to a pH of 6.8 with 10% sodium hydroxide solution, heated to 60 ° C. for 10 hours and then immediately cooled in ice water and filtered by filtration through a membrane filter with a pore size of 0.45 u. sterilized. Each 1 ml of the sterilized solution obtained is filled aseptically into glass ampoules which have been sterilized at 180 ° C for 2 hours beforehand. After freeze-drying under aseptic conditions, the ampoules are melted down. 97 ampoules, each containing 1 mg of a preparation containing heat-treated HGI glycoproteid, are obtained.

The preparations obtained are examined for their biological activity and viral infectivity according to experiment 5. The biological activity is comparable to that of an untreated sample and no viral infectivity can be determined.

Example 5

1000 liters of fresh urine from healthy people is treated according to Example 1. The elution liquid from the 40 CM Sephadex C-50 column is concentrated in 1 liter of crude HGI glyco-proteid solution. The raw concentrate is mixed with 10 liters of a 0.1M tris-HCl buffer solution with a pH of 7.0, stirred thoroughly and again concentrated to about 1 liter using a DIAFLO hollow fiber ultrafiltration device. The concentrate is mixed with 5 liters of 0.1M tris-HCl buffer solution with a pH of 7.0 and 5 liters of a DEAE-cellulose suspension (300 g DEAE-cellulose on a dry basis), previously with 0.1M tris-HCl -Buffer solution has been equilibrated to pH 7.0. The mixture is stirred for 30 to 50 minutes and, after standing, is filtered under reduced pressure. The filtered DEAE cellulose is washed with 10 liters of a 0.1M tris-HCl buffer solution with a pH of 7.0. The DEAE cellulose is then filtered off under reduced pressure and washed again with 10 liters of a 55 0.1M tris-HCl buffer solution which is 0.05M sodium chloride. The DEAE cellulose is then filtered off under reduced pressure. The filtered DEAE cellulose is mixed with 10 liters of a 0.1M tris-HCl buffer solution of pH 7.0, which is 0.3M sodium chloride, and 60 is stirred to elute the HGI glycoproteide fraction. The eluate is desalted using a DIAFLO hollow fiber ultrafiltration device (type DC-30). The desalted fraction is freeze-dried. About 15 g of a powder are obtained. This powder is dissolved in 150 ml of distilled water and placed on a column of 6.0 x 80 cm filled with Sephadex G-150, which has been equilibrated with 0.1 M tris-HCl buffer solution with a pH of 7.0 is. The fractions correspond to a Ve / Vo ratio of 1.11 to 1.60 and are collected. The fractions are pooled and dialyzed thoroughly against distilled water. The dialyzed solution (retentate) is then concentrated using a DIAFLO hollow fiber ultrafiltration device (type DC-2). 100 ml of a concentrate are obtained, which contains about 3 g of crude HGI glycoproteide.

10 g of powdered human placenta albumin (produced according to the example of JA-AS 40 132/76) with a purity of 98% (determined by electrophoresis) are dissolved in the concentrate obtained. The solution obtained is adjusted to a pH of 6.8 with 10% sodium hydroxide solution, sterilized by filtration according to Example 4, filled aseptically in 2.5 ml portions into ampoules and aseptically freeze-dried. The ampoules are then melted down. 40 ampoules, each with 3.8 mg of heat-treated HGI glycoproteid, are obtained. The preparations obtained are examined for biological activity and viral infectivity according to experiment 5. The biological activity is comparable to the non-heat-treated preparations and no viral infectivity can be determined.

Example 6

400 liters of fresh urine from healthy people are adjusted to a pH of 8 in 10% sodium hydroxide solution and then centrifuged with cooling at 10 ° C. in a continuous centrifuge at 15,000 × g to remove insoluble substances. The supernatant obtained is adjusted to pH 7 with 10% hydrochloric acid and placed on a column filled with silica gel with the dimensions 10 × 80 cm. The substances adsorbed on the silica gel are eluted with 40 liters of 5 percent ammonia solution. The eluate obtained is adjusted to pH 7.5 with sulfuric acid and ammonium sulfate is added to a saturation level of up to 70 percent. After standing at 0 ° C. for 15 to 18 hours, the precipitate formed is filtered off. The precipitate obtained is dissolved in 2 liters of 5 percent ammonia solution, filled into cellophane tubes and dialyzed thoroughly against 0.05M phosphate buffer with a pH of 6.5. The dialyzed solution obtained (the retentate) is made up to 10 liters with the same buffer and placed on a 40 × 40 cm column filled with Sephadex C-50, which has been equilibrated with 0.05 M phosphate buffer with a pH of 6.5 . Contamination is separated by adsorption. 10 liters of the eluate obtained are concentrated using a DIAFLO hollow fiber ultrafiltration device (Amicon, type DC-30). The concentrate obtained is dialyzed against 0.1 M tris-HCl buffer solution with a pH of 7.0 to 5 ° C. for 15 to 18 hours in the manner described above.

The dialyzed solution obtained (the retentate) is made up to 1 liter with the same buffer solution and onto a DEAE cellulose column with the dimensions 4.0 × 40 cm

640865

14

set which has been equilibrated with the same buffer solution. The column is washed thoroughly with 0.1 M tris-HCl buffer solution of pH 7.0 and then eluted with a 0.1 M tris-HCl buffer solution of pH 7.0 containing 0.3 M sodium chloride is. Those fractions which stimulate the proliferation and differentiation of mouse bone marrow cells in vitro (determined according to experiment 5) are collected. The fractions are pooled and dialyzed against 0.1M tris-HCl buffer solution with pH 7.0. The dialyzed solution (the retentate) is again placed on a DEAE cellulose column measuring 4.0 × 40 cm, which has been equilibrated with the same buffer solution. The column is eluted with a linear concentration gradient of a saline solution (0 to 0.3 M). The fractions with a stimulating effect on the proliferation and differentiation of granulocytes are collected and treated with ammonium sulfate up to 70 percent saturation. The resulting precipitate is filtered off, dissolved in a little 0.1M tris-HCl buffer solution with a pH of 7.0 and dialyzed against the same buffer solution.

20 ml of the dialyzed solution obtained (retentate)

are placed on a Sephadex G-150 column with the dimensions 4.0x60 cm, which has been equilibrated with a 0.1 M tris-HCl buffer solution with a pH of 7.0. The eluate fractions with a Ve / Vo ratio of 1.11 to 1.45 are collected. The fractions are pooled and dialyzed thoroughly against distilled water. The dialyzed solution (the retentate) is then freeze-dried. About 500 mg of a powder are obtained.

200 mg of the powder obtained are dissolved in 0.02M phosphate buffer solution of pH 7.0, which is 1.0M in sodium chloride, and placed on a column containing 100 ml of Con-canavalin A-Sepharose 4B, the same Buffer solution has been equilibrated. The column is thoroughly washed with 0.02M phosphate buffer solution, pH 7.0, which is 1.0M sodium chloride, and then with a 0.02M phosphate buffer solution, pH 7.0, which contains 50mM a-methyl-D -glucoside and 1.0mM sodium chloride, eluted. The fractions with a stimulating effect on the proliferation and differentiation of the granulocytes (determined according to experiment 5) are collected and dialyzed against distilled water. The dialyzed solution is freeze-dried.

About 50 mg of the freeze-dried powder obtained are dissolved in 1 ml of a 0.125 M tris-glycocoll buffer solution of pH 6.8, which contains 10% glycerol. The solution obtained is subjected to electrophoresis at 10 mA with cooling using a preparative electrophoresis device (Fuji Kabara II type) using 8% acrylamide gel (pH 8.9; 20 mm × 25 mm). The fraction with a relative mobility of 0.46 is eluted with 0.025 M tris-Glycolkoll buffer solution with a pH of 8.3 and then dialyzed against distilled water. The dialyzed solution is freeze-dried. About 10 mg of HGI glycoproteid are obtained.

The procedure described above is repeated several times and about 1 g of HGI glycoproteid is obtained. 1 g of the HGI glycoprotein obtained is completely dissolved in 10 ml of water and adjusted to a pH of 6.8 with 10% sodium hydroxide solution. The solution obtained is then heated to 60 ° C. for 10 hours, then rapidly cooled in ice water and diluted to 10 times its volume with sterile water. The solution is then sterilized by filtration through membrane filters with a pore size of 0.45 (i. 1 ml of the sterilized solution obtained is filled aseptically into glass ampoules which have been heated for 2 hours at 180 ° C. After aseptic freeze-drying, the glass ampoules are melted off About 97 ampoules, each containing 10 mg of heat-treated HGI glycoproteid, are obtained.

The preparations obtained are examined for biological activity and viral infectivity according to experiments 5 and 7. The biological activity is about 40% that of a preparation before the heat treatment, and no viral activity can be detected.

Example 7

1000 liters of fresh urine from healthy people is treated according to Example 6. 2.5 liters of an aqueous solution which contains crude HGI glycoproteid are obtained from the eluate of the CM Sephadex C-50 column. 25 liters of a 0.1M tris-HCl buffer solution with a pH of 7.0 are added to the solution. After thorough stirring, the solution is concentrated using a DIAFLO hollow fiber ultrafiltration device to about one-half its initial volume. The concentrate is mixed with 5 liters of a 0.1M tris-HCl buffer solution and 5 liters of a DEAE cellulose suspension (300 g DEAE cellulose on a dry basis), which is mixed with 0.1 M tris-HCl buffer solution with a pH of 7. 0 has been equilibrated. The mixture is stirred for 30 minutes, then left to stand and then filtered off under reduced pressure. The DEAE cellulose filtered off is washed with 10 liters of a 0.1 M tris-HCl buffer solution of pH 7.0 and filtered off under reduced pressure. The DEAE cellulose is then washed with 0.1M tris-HCl buffer solution of pH 7.0, which is 0.05M sodium chloride, and then filtered off again under reduced pressure. The DEAE cellulose treated in this way is stirred in 10 liters of a 0.1M tris-HCl buffer solution of pH 7.0, which is 0.3M sodium chloride, in order to elute the HGI glycoproteid fraction. The eluate is desalted using a DIAFLO hollow fiber ultrafiltration device (type DC-30). After freeze-drying, about 15 g of a powder are obtained.

The freeze-dried powder is dissolved in 150 ml of distilled water and placed on a Sephadex G-150 column with the dimensions 6.0 x 80 cm, which has been equilibrated with 0.1M tris-HCl buffer solution with a pH of 7.0. The HGI glycoprotein fractions with a Ve / Vo ratio of 1.11 to 1.60 are collected and dialyzed thoroughly against distilled water.

The dialyzed solution obtained (the retentate) is concentrated with a DIAFLO hollow fiber ultrafiltration device (type DC-2). 30 ml of a concentrate are obtained, which contains about 3 g of crude HGI glycoproteide. The concentrate is adjusted to a pH of 6.8 with sodium hydroxide solution and heat-treated according to Example 6. The heat-treated concentrate obtained is sterilized by filtration and aseptically filled into 1.0 ml portions in ampoules. After aseptic freeze drying, the ampoules are hermetically sealed. 30 ampoules, each containing 5.2 mg of heat-treated HGI glycoproteid, are obtained.

The preparations obtained are examined for biological activity and viral infectivity according to experiments 5 and 7. The biological activity is about 40% of that before the heat treatment, and no viral activity can be detected.

Example 8

The procedure of Example 1 is repeated. About 500 mg (about 26 mg of active ingredient) of HGI glycoproteid powder corresponding to sample No. 4 are obtained, but the following stage is switched on before the stage of dialysis of the 5 percent aqueous ammonia solution of ammonium sulfate precipitation: About 20 g of the ammonium sulfate precipitation obtained see

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are dissolved in 200 ml of water (protein concentration 100 mg per ml). The solution obtained is heated to 60 ° C. for 10 hours and then rapidly cooled in ice water. A fraction is obtained which contains the heat-treated HGI glycoproteid. This fraction is dialyzed according to example 1. The biological activity of the HGI-Gly-coproteid powder obtained is comparable to that of the fraction before the heat treatment, and no viral activity can be determined.

Example 9

About 450 mg of a powder corresponding to sample No. 4 of Example 1, which contain 23.5 mg of HGI glycoproteid, are produced according to Example 1, but with the following modification:

10 liters of the eluate from the CM Sephadex C-50 column are concentrated using a DIAFLO hollow fiber ultrafiltration device up to a protein concentration of 70 mg per ml. The concentrated aqueous solution is heated to 60 ° C. for 10 hours and then rapidly in ice water cooled down. Insoluble substances are filtered off, and the aqueous solution is filled into cellophane tubes and dialyzed against 0.1 M tris-HCl buffer solution of pH 7.0 at 5 ° C. The dialyzed solution (the retentate) is made up to 1 liter with the same buffer solution. A solution corresponding to sample No. 1 from example 1 is obtained.

The biological activity of the HGI glycoprotein powder obtained is comparable to that of the powder obtained without heat treatment. No viral activity can be detected.

Example 10

About 500 mg of a powder corresponding to sample No. 4 from Example 1, which contain about 26 mg of HGI glycoproteid, are prepared according to Example 1, but with the following modification:

The eluate from the DEAE cellulose column is precipitated with ammonium sulfate. The precipitate obtained is dissolved in a 0.1M tris-HCl buffer solution up to a protein content of 150 mg per ml. The solution obtained is heated to 60 ° C. for 10 hours and then quickly cooled in ice water. The resulting precipitate is filtered off and the filtrate is dialyzed against 0.1 M tris-HCl buffer solution. The dialyzed solution obtained is used in place of Sample No. 3 in Example 1. The biological activity of the HGI glycoproteid powder finally obtained is comparable to that of the preparation which was obtained without heat treatment. No viral activity can be detected.

Example 11

50 liters of fresh urine from healthy people is neutralized with 10% sodium hydroxide solution and centrifuged at 10,000 x g using a coolable continuous centrifuge. s The supernatant is concentrated to its volume by means of a DIAFLO hollow fiber ultrafiltration device (Aminon Co., type DC-30, equipped with a membrane that cuts off a molecular weight of 10,000). After adding 50 liters of water, the solution is concentrated again. 3 liters of concentrate are obtained. The concentrated urine is evaporated using a rotary evaporator to about 50 ml of concentrate with a protein content of 70 mg per ml. The concentrate is heated to 60 ± 0.5 ° C for 10 hours and then cooled rapidly. A heat-treated fraction containing HGI glycoprote is obtained. After the removal of insoluble substances, the concentrate is filled with 0.1M tris-HCl buffer solution from pH 7.0 to 5 liters and placed on a DEAE cellulose column with the dimensions 4.0x40 cm, which was previously filled with 20 0.1M tris -HCl buffer solution has been equilibrated. The HGI glycoprotein is adsorbed on the DEAE cellulose. The column is washed thoroughly with 0.1 M tris-HCl buffer solution, pH 7.0, and eluted with 0.1 M tris-HCl buffer solution, pH 7.0, containing 0.3 M of natri-25 umchloride is. The process according to Example 1 is then repeated, but the eluate obtained is used instead of the 5 percent aqueous ammonia solution of the 70 percent ammonium sulfate precipitation precipitate. About 200 mg of HGI glycoproteid powder corresponding to 30 Sample No. 4 are obtained after repeating the procedure of Example 1.

The biological activity of the HGI glycoprotein powder obtained is comparable to that of the preparation obtained without heat treatment. No viral activity can be detected.

Example 12

The procedure of Example 1 is repeated, a fraction corresponding to Sample No. 3 from Example 1 is isolated. 40 This fraction is concentrated to a protein content of 70 mg per ml and heated at 60 ° C for 10 hours. The fraction is then rapidly cooled in ice water, the precipitate formed is removed and further treated in the same manner as in Example 1. About 10 mg of an HGI-45 glycoproteid powder corresponding to sample No. 6 from Example 1 are obtained.

The biological activity of the HGI glycoproteid powder obtained is comparable to that of the preparation without heat treatment.

2 sheets of drawings

Claims (25)

640 865 2nd PATENT CLAIMS 1. Glycoproteid of human urine with a molecular weight of 75,000 to 90,000 u determined by gel filtration with the following physical and chemical parameters: (a) Solubility: Soluble in water, somewhat soluble in chloroform and insoluble in ethanol and acetone; (b) specific rotation: [a] o = 0 ± 40 ° at c = 0.25% in water; (c) pH: 5.0 to 6.0 1 weight percent aqueous solution; (d) Isoelectric point: pH 4.7 ± 0.2; (e) Thermostability: complete loss of the stimulatory effect on the proliferation and differentiation of human granulocytes after heating a 1% aqueous solution at 60 ± 0.5 ° C for 30 minutes; (f) Electrophoresis: Relative mobility 0.25 in electrophoresis on sodium dodecyl sulfate-polyacrylamide gel; (g) IR absorption spectrum: characteristic absorption at 3600 to 3200 strong absorption, 1700 to 1600 strong absorption, 1550 medium absorption, 1430 to 1380 (medium absorption) and 1150 to 1000 broad band cm-1; (h) Color reactions: characteristic color reaction for saccharides with a-naphthol-sulfuric acid, indole-sulfuric acid, anthrone-sulfuric acid and phenol-sulfuric acid; characteristic color reaction for polypeptide bonds and amino acids according to the Lo wry method and the Ninhy-drin reaction after hydrolysis with hydrochloric acid; (i) Basic amino acid building blocks of the protein residue: proline, aspartic acid, threonine, serine, glutamic acid, glycocoll, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, tryptophan and arginine; (j) color and crystal shape: white and amorphous; (k) Sugar composition of the polysaccharide residue: 10.0 to 13.0 percent by weight of neutral sugars expressed in glucose, 3.0 to 7.0 percent by weight of sialic acids and 1 percent by weight of other aminosugars; (1) Weight ratio of protein to polysaccharide: 75 to 85: 13.0 to 20.0; (m) Elemental analysis: 42.3 to 47.3% carbon, 5.7 to 7.8% hydrogen, 9.6 to 14.3% nitrogen, 34.4 to 39.4% oxygen and at most 0.2% Sulfur. 2. Process for the preparation of the glycoprotein according to Claim 1, characterized in that the proteins contained in human urine are concentrated, the proteins obtained are treated with a cation exchanger for the separation of impurities, the eluate for the adsorption of the glycoprotein is treated with an anion exchanger which Glycoproteid from the anion exchanger eluted with a saline solution with a linear concentration gradient, the eluate being subjected to gel filtration chromatography on a strongly crosslinked polymer gel to develop the glycoproteid, and the fractions with realistic elution volumes Ve / Vo from 1.11 to 1.60 catches. 3. The method according to claim 2, characterized in that the collected fractions are additionally subjected to affinity chromatography on an absorbent with affinity for sugar, and the adsorbed glycoproteid is eluted with a 20 to 100 mmol saccharide solution. 4. The method according to claim 3, characterized in that the eluate is subjected to preparative zone electrophoresis and the glycoproteid is eluted with a salt solution. 5. The method according to claim 2 to 4, characterized in that the relative elution volume is 1.11 to 1.45. 6. The method according to claim 2, characterized in that the fractions are adjusted to a pH of 5 to 9 with a relative elution volume of 1.11 to 1.60 and the aqueous solution obtained for inactivating viruses in the presence of human albumin Heated to 50 to 70 ° C for 8 to 30 hours. 7. The method according to claim 6, characterized in that the albumin concentration in the aqueous solution is at least 2.0% by weight per volume. 8. The method according to claim 6, characterized in that the albumin comes from human serum or human placenta. 9. The method according to claim 2, characterized in that the fractions with a relative elution volume of 1.11 to 1.60 are subjected to affinity chromatography on an absorbent with affinity for sugar, the adsorbed glycoproteid is eluted with a 20 to 100 mmol saccharide solution, the eluate obtained is adjusted to a pH of 5 to 9 and the aqueous solution obtained for inactivating viruses is heated to 50 to 70 ° C. in the presence of human albumin for 8 to 30 hours. 10. The method according to claim 9, characterized in that the albumin concentration in the aqueous solution is at least 2.0% weight by volume. 11. The method according to claim 9, characterized in that the albumin comes from human serum or human placenta. 12. The method according to claim 2, characterized in that the fractions are treated with a relative elution volume of 1.11 to 1.60 of affinity chromatography on an absorbent with affinity for sugar, the adsorbed glycoproteid is eluted with a 20 to 100 mmol saccharide solution, the eluate obtained is subjected to preparative zone electrophoresis, the glycoproteid is eluted with a saline solution, the eluate obtained is adjusted to a pH of 5 to 9 and heated to 50 to 70 ° C. for 8 to 30 hours in order to inactivate viruses in the presence of human albumin. 13. The method according to claim 12, characterized in that the albumin concentration in the aqueous solution is at least 2.0% by weight per volume. 14. The method according to claim 12, characterized in that the albumin comes from human serum or human placenta. 15. The method according to claim 2, characterized in that the fractions with a relative elution volume of 1.11 to 1.60 are subjected to affinity chromatography on an absorbent with affinity for sugar, the adsorbed glycoproteid is eluted with a 20 to 100 mmol saccharide solution, subjecting the eluate to preparative zone electrophoresis, eluting the glycoproteid with a saline solution, adjusting the concentration of the glycoproteid to at least 70 mg per ml and the pH value to 5 to 9 in the eluate and the resulting eluate to 50 for 8 to 30 hours for inactivating viruses heated to 70 ° C. 16. The method according to claim 15, characterized in that the concentration of the glycoprotein at the virus inactivation stage is 100 to 200 mg per ml. 17. The method according to claim 2, characterized in that the fractions containing the glycoproteid, including the protein-containing concentrated human urine, for inactivating viruses in any stage in the form of an aqueous solution at 50 to 70 ° C for 8 to 30 hours heated such conditions that the protein content of the aqueous solution has been adjusted to at least 70 mg per ml and the pH to 5 to 9. 18. The method according to claim 17, characterized in that the protein content of the aqueous solution for virus inactivation is 100 to 150 mg per ml. 5 10th 15 20th 25th 30th 35 40 45 50 55 60 65 3rd 640 865 19. The method according to claim 2, characterized in that the fractions with a relative elution volume of 1.11 to 1.60 are subjected to affinity chromatography on an absorbent with affinity for sugar and the adsorbed glycoproteid is eluted with a 20 to 100 mmol saccharide solution, wherein the fractions containing the glycoprotein, including the protein-containing concentrated human urine, are inactivated in any stage in the form of an aqueous solution at 50 to 70 ° C for 8 to 30 hours at 50 to 70 ° C under conditions such that the protein content of the aqueous solution has been adjusted to a value of at least 70 mg per ml and the pH to 5 to 9. 20. The method according to claim 19, characterized in that the protein content of the aqueous solution is 100 to 150 mg per ml. 21. The method according to claim 2, characterized in that the fractions with a relative elution volume of 1.11 to 1.60 are subjected to affinity chromatography on an absorbent with affinity for sugar, the adsorbed glycoproteid is eluted with a 20 to 100 mmol saccharide solution, subjecting the eluate to preparative zone electrophoresis and eluting the glycoproteid in pure form with a saline solution, taking the fractions containing the glycoproteid, including the concentrated human urine containing proteins, to inactivate viruses at any stage in the form of an aqueous solution for 8 to 30 hours heated to 50 to 70 ° C under conditions such that the protein content of the aqueous solution has been adjusted to at least 70 mg per ml and the pH to 5 to 9. 22. The method according to claim 21, characterized in that the protein content of the aqueous solution for virus inactivation is 100 to 150 mg per ml. 23. Medicament for combating leukocytopenia, characterized by a content of the glycoproteid according to claim 1. 24. Medicament according to claim 23, characterized in that it is free from viral infectivity. 25. Medicament according to one of claims 23 or 24, characterized by a content of the glycoproteid with a specific biological activity of at least 35,000 units per mg.